ZooGene is an international partnership to create a database
of DNA type sequences for calanoid copepods and euphausiids (see http://www.ZooGene.org).ZooGene has recently expanded to
include gelatinous zooplankton groups.Following taxonomic identification of specimens, a DNA type sequence is
determined for a 660-base pair portion of the mitochondrial cytochrome oxidase
I (mtCOI) gene; multiple mtCOI sequences are included as necessary to reflect
intraspecific variation.Different
genes are used for some groups or species, as needed.

Samples collected with
minimum damage to the specimens are preferred (i.e., short tows taken in good
weather).Samples must be
preserved immediately upon collection.Only those individuals that are ALIVE up to the moment of preservation
should be used.DNA is destroyed
by enzymes immediately upon the death of the organism.

Crustacean
zooplankton: The ZooGene
project seeks zooplankton collections containing calanoid copepods and
euphausiids from any region of the world oceans; samples must be preserved
especially for molecular analysis following the instructions provided
here.In most cases, we would
greatly appreciate your assistance in providing specimens identified to
species, with the identification confirmed by a taxonomic authority.In this case, please send 20 to 30
individuals (adult females are preferred) in a small glass vial (5 - 20 ml)
with a plastic top that will not leak or allow evaporation.Please include a label inside the vial;
use a label without ink markings and write all collection information in
pencil.

Gelatinous
zooplankton: ZooGene has
recently expanded its taxonomic range to include gelatinous zooplankton groups,
and we are focusing on ctenophores, planktonic cnidarians, and larvaceans,
among others.Please let us know
of any samples or specimens you may be able to collect, and we can determine
which ones are of most use for us.Generally, 5 to 10 individuals per species are desired.Assuming the taxonomic
identification of the specimen is definitive, the size and maturity of the
specimen does not matter.If
collections are made from diverse regions, we prefer to have specimens a given
species from a selection of sites, but this is not necessary.

PROTOCOLS FOR COLLECTION
AND PRESERVATION OF ZooGene SAMPLES

Please follow these
instructions carefully.Unless
samples are carefully preserved, specimens will yield no DNA for our
analyses.This protocol is also
available on the project website at http://www.ZooGene.org.

Steps 1 – 3 for
Crustacean zooplankton

1)Samples should be collected using nets with 100 um to 333 um mesh
for copepods, and 333 to 550 um mesh for euphausiids.Samples need not be quantitative; non-quantitative portions
of samples are acceptable.

2)Immediately after collection, drain samples of excess seawater
(using a sieve with mesh of same size - or smaller - as the net).

3)Wash the sample into a glass jar using 95% un-denatured (i.e.,
drinkable) ethyl alcohol.Add
additional 95% ethyl alcohol to fill the jar.NOTE: there must be 3 to 4 times more alcohol than plankton
volume.Samples can be split to
keep plankton biovolume to one-third or one-fourth of the jar volume.We recommend removing fish from the
samples.

Steps 1 – 3
forGelatinous zooplankton

1)Some gelatinous zooplankton may be collected in nets; others will
require special collection methods (submersible, ROV, divers).Samples need not be quantitative;
non-quantitative portions of samples are acceptable.

2)For very large
individuals, tissue can be excised prior to preservation.Whether entire individuals or excised
tissue is preserved, different vials should be used for each individual (or
colony).If portions of animals
are preserved, care should be taken to avoid non-cellular regions (e.g., inner
bell matrix).Wash the sample into a glass jar using 95% un-denatured (i.e.,
drinkable) ethyl alcohol.Add
additional 95% ethyl alcohol to fill the jar.NOTE: there must be 3 to 4 times more alcohol than plankton
volume.

3)Gelatinous
zooplankton specimens that disintegrate in alcohol can still be used for
molecular analysis.If the
specimen has disintegrated, DO NOT change the alcohol, since the DNA will have
dissolved or remained in flocculent material.Note that gelatinous specimens can also be placed in
cryovials and flash frozen in liquid nitrogen.Label the cryovials with an indelible felt-tip pen,
including species name, collection date, and geo-reference coordinates.

Steps 4 – 7 for all
ZooGene samples

4)NOTE:ONLY 95%
UNDENATURED ETHYL ALCOHOL CAN BE USED TO PRESERVE ZOOGENE SAMPLES.PLEASE DO NOT USE 100%
ETHANOL.DO NOT USE DENATURED
ALCOHOL.If you are not sure which
alcohol to use, please ask us for specific information and suggestions for
vendors in your region.

5)Place a label inside the jar or vial, writing in pencil.Unprinted labels are preferred since
the ink may dissolve in the alcohol.Use small labels made from acid-free paper.(We have discovered that some labels change the sample pH
significantly, especially in small volumes).Sample pH should remain close to pH 8.0.Please note collection information
desired: cruise (ship and cruise name or number); collection date and local
time; georeference coordinates (latitude and longitude); station or tow number;
net and mesh size.

6)After 24 hours, drain off alcohol and replace with fresh
alcohol.Continue to change the
alcohol every 24 to 48 hours, until the fluid remains clear and free of debris.

7)Ship samples to Ann Bucklin. Follow the Guidelines for the
Shipment of Excepted Quantities of Flammable Liquids. DO NOT ship via airmail;
use a carrier that accepts hazardous materials such as DHL, FedEx, etc. Costs
of sample shipment will be paid or reimbursed upon request.

MICROSCOPIC EXAMINATION
OF SPECIMENS FOR MOLECULAR ANALYSIS

When microscopic
examination is required for species identification of copepods, euphausiids, or
gelatinous forms, please take special care in order to allow later molecular
analysis of these individuals.View the specimens in 95% ethyl alcohol; do not move them to water or
other fluid.Do not use stains or
other treatments.Avoid
dissection; if necessary, use sterilized tools that have never been exposed to
formalin; do not use the same tools for multiple individuals.Do not allow the alcohol to evaporate
or become warm; minimize light exposure by limiting both duration and
intensity.

If such handling is not
possible to allow identification, consider examining only some individuals of
each sample carefully.Keep these
and send the others individuals of that sample to us for molecular analysis.

THANK YOU VERY MUCH!

Please don’t hesitate to
contact me if you have any questions or concerns about collection, preservation,
and shipment of ZooGene samples.

Ann Bucklin

Professor of Zoology /
EOS

Ocean Process Analysis
Laboratory

University of New
Hampshire

Durham, NH 03824 USA

Tel. (603) 862-0122; Fax
(603) 862-0243; Email ann.bucklin@unh.edu

Guidelines for the Shipment of Excepted Quantities of Flammable Liquids

International
and domestic hazardous material shipping regulations protect individuals and
property involved in the shipment of hazardous materials. Shipments of
flammable liquids may be made according to the Excepted Quantity provision of
the International Air Transport Association (IATA) and U.S. Department of
Transportation (DOT) regulations. Shipments made under this exception
must only be made by a person trained to use IATA Dangerous Goods Regulations
and/or U.S. DOT Hazardous Material Regulations.

Note:
flammable liquids are not permitted in checked baggage, carry-on baggage or
airmail.

Packaging

Packaging
for excepted quantities of flammable liquids must have three basic components:

1.Inner packaging, such
as a vial, tube, jar, etc. Do not completely fill inner packagings; allow space
for liquid expansion. Liquids must not completely fill inner packagings at a
temperature of 55ºC (130ºF). Closures of inner packagings must be held securely
in place with tape, wire, metal crimps or other positive means.

2.Intermediate packaging,
such as a ziplock or other plastic bag. Intermediate packaging must contain
enough absorbent material to absorb all contents.

3.Outer packaging, such
as a cardboard (fibreboard) box. Flammable liquids may not be shipped in
envelopes, Tyvek® sleaves, or other non-rigid mailers. The dimensions of the
outer box must be at least 100 mm on two of three sides.

Quantity Limits

95%
ethanol is a flammable liquid in hazard class 3, packing group II. As such,
each inner packaging may contain not more than 30 mL. Each outer package may
contain not more than 500 mL.

Example: If you are shipping 10 mL vials of 95%
ethanol, you may put up to 50 vials in one box.

Documentation

The
“Nature and Quantity of Goods” box of the air waybill must include the words
“Dangerous Goods in Excepted Quantities.” See attached DHL air waybill.

Labeling

An
Excepted Quantities of Dangerous Goods Label must be completely filled out and
affixed to the outer container. For 95% ethanol check the box for Class 3
material and write the UN number UN 1170. The attached label may be used, but
it must be printed in color. Consider printing it on an adhesive backed label.
Its overall dimensions must be at least 100mm X 100mm.

For a copy of the DHL Shipment Air Waybill paperwork on a separate page,
click here.